Automated contamination-free seed sampler and methods of sampling, testing and bulking seeds

ABSTRACT

An automated seed sampler system includes an imaging device for obtaining images of seeds, an orienting device for orienting the seeds based on the images, and a sampling station for removing tissue from the oriented seeds. In some aspects, the system also includes a transport subsystem for supporting the oriented seeds and conveying the oriented seeds to the sampling station. A method for removing tissue from seeds includes imaging the seeds, orienting the seeds based on image information obtained from the seeds, and removing tissue from the oriented seeds. In some aspects, the method also includes transporting the oriented seeds in a transport subsystem to a sampling station for removing the tissue from the oriented seeds, and/or collecting the tissue removed from the oriented seeds so that a one-to-one correspondence exists between the tissue and the sampled seeds, and/or analyzing the tissue for characteristics indicative of genetic and/or chemical traits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/210,212, filed Aug. 15, 2011 (now U.S. Pat. No. 8,539,713), which isa divisional of U.S. patent application Ser. No. 11/680,180, filed Feb.28, 2007 (now U.S. Pat. No. 7,998,669), which claims priority to and thebenefit of U.S. Provisional Application No. 60/778,830, filed Mar. 2,2006. The disclosures of each of these applications are incorporatedherein by reference in their entireties.

FIELD

This disclosure relates to systems and methods for taking samples frombiological materials such as seeds.

BACKGROUND

The statements in this section merely provide background informationrelated to the present disclosure and may not constitute prior art.

In plant development and improvement, genetic improvements are made inthe plant, either through selective breeding or genetic manipulation,and when a desirable improvement is achieved, a commercial quantity isdeveloped by planting and harvesting seeds over several generations. Notall seeds express the desired traits, and thus these seeds need to beculled from the population. To speed up the process of bulking up thepopulation, statistical samples are taken and tested to cull seeds fromthe population that do not adequately express the desired trait.However, this statistical sampling necessarily allows some seeds withoutthe desirable trait to remain in the population, and also caninadvertently exclude some seeds with the desirable trait from thedesired population.

U.S. patent application Ser. No. 11/213,430 (filed Aug. 26, 2005); U.S.patent application Ser. No. 11/213,431 (filed Aug. 26, 2005); U.S.patent application Ser. No. 11/213,432 (filed Aug. 26, 2005); U.S.patent application Ser. No. 11/213,434 (filed Aug. 26, 2005); and U.S.patent application Ser. No. 11/213,435 (filed Aug. 26, 2005), which areincorporated herein by reference in their entirety, disclose apparatusand systems for the automated sampling of seeds as well as methods ofsampling, testing and bulking seeds.

However, at least some known automated sampling and testing systemsallow for various types of contamination to taint collected samples andskew results. Therefore, there exists a need for the automated samplingof seeds in a substantially contamination-free manner.

SUMMARY

The present disclosure relates to systems and methods ofnon-destructively sampling material from seeds. The methods areparticularly adapted for automation, which permits greater sampling thanwas previously practical. With automated, non-destructive samplingpermitted by at least some of the embodiments of this disclosure, it ispossible to test every seed in the population, and cull those seeds thatdo not express a desired trait. This greatly speeds up the process ofbulking a given seed population, and can result in an improved finalpopulation.

Various embodiments of the present disclosure facilitate the testing ofmost or all of the seeds in a population before planting, so that timeand resources are not wasted in growing plants without the desiredtraits. Further, various embodiments allow for the automated sampling ofseeds in a contamination-free manner, thereby substantially eliminatingcross-over between samples.

In various embodiments, the present disclosure provides an automatedseed sampler system that includes a milling station for removing atleast a portion of seed coat material from a seed and a sampling stationfor extracting a sample of seed material from the seed where the seedcoat has been removed. A seed transport subsystem conveys the seedbetween the milling station and the sampling station and a seed depositsubsystem conveys the seed from the seed transport subsystem to aselected well in a seed tray after the seed has been sampled.

In various other embodiments, the present disclosure provides anautomated seed sampler system that includes a milling station forremoving at least a portion of seed coat material from a seed and asampling station for extracting a sample of seed material from the seedwhere the seed coat has been removed. A sample collection and transportsubsystem captures the extracted sample in a collection tube mounted ona collection tube placement device of the sample collection andtransport subsystem. Additionally, a sample deposit subsystem conveysthe sample from the sample collection and transport subsystem to aselected well in a sample tray.

In yet other various embodiments, the present disclosure provides amethod of extracting sample material from a seed for testing. The methodincludes loading a seed in a seed holder of an automated seed samplersystem and removing at least a portion of seed coat material from theseed at a milling station of the seed sampler system. A sample of seedmaterial is then extracted from the seed where the seed coat has beenremoved at a sampling station of the seed sampler system. The sampledseed is then conveyed to a selected well in a seed tray using a seeddeposit subsystem of the seed sampler system. The extracted sample iscoincidentally conveyed to a selected well in a sample tray using asample deposit subsystem of the seed sampler system. The depositedsample can then be tested for at least one desired seed characteristic.

In still other embodiments, the present disclosure provides an automatedsystem for sequentially removing sample material from a plurality ofseeds while leaving the viability of the seeds intact. The systemincludes a milling station for sequentially removing at least a portionof seed coat material from each seed and a sampling station forsequentially extracting a sample of seed material from each seed wherethe seed coat has been removed from the respective seed. A seedtransport subsystem conveys the seeds between the milling station andthe sampling station and a seed deposit subsystem sequentially conveyseach seed from the seed transport subsystem to a selected one of aplurality of wells in a selected one of a plurality of seed trays. Thesystem additionally includes a sample collection and transport subsystemfor sequentially capturing the extracted sample of each seed in acorresponding collection tube mounted on one of a plurality ofcollection tube placement devices. The system further includes a sampledeposit subsystem for sequentially conveying each sample from the samplecollection and transport subsystem to a selected one of a plurality ofwells in a selected one of a plurality of sample trays.

In other embodiments of the present disclosure, a method for removingtissue samples from seeds generally includes orienting seeds in adesired orientation, transporting the oriented seeds to a samplingstation, and removing tissue samples from the oriented seeds at thesampling station.

In other embodiments of the present disclosure, an automated method forremoving a tissue sample from a seed generally includes isolating anindividual seed from a plurality of seeds, orienting the isolated seedusing an actuator, and removing a tissue sample from the oriented seed.Here, the actuator is configured to position the seed in a desiredorientation.

In other embodiments of the present disclosure, a method for removingtissue samples from seeds generally includes orienting multiple seedstogether in a seed transport and removing tissue samples from theoriented seeds while the oriented seeds are in the seed transport.

The systems and methods of this disclosure facilitate the automated,non-destructive sampling of seeds in a substantially contamination-freemanner. They permit the testing and sorting of large volumes of seeds,thereby facilitating the bulking up of seed populations with desirabletraits. These and other features and advantages will be in partapparent, and in part pointed out hereinafter.

Further areas of applicability of the present teachings will becomeapparent from the description provided herein. It should be understoodthat the description and specific examples are intended for purposes ofillustration only and are not intended to limit the scope of the presentteachings.

DRAWINGS

The drawings described herein are for illustration purposes only and arenot intended to limit the scope of the present teachings in any way.

FIG. 1 is a perspective view of a seed sampler system in accordance withvarious embodiments of the present disclosure.

FIG. 2 is an enlarged perspective view of a seed loading station of theseed sampler system shown in FIG. 1, in accordance with variousembodiments of the present disclosure.

FIG. 3 is an enlarged perspective view of a seed orientation system ofthe seed sampler system shown in FIG. 1, in accordance with variousembodiments of the present disclosure.

FIG. 4 is a side elevation view of the seed orientation system shown inFIG. 3, in accordance with various embodiments of the presentdisclosure.

FIG. 5 is a perspective view of the seed orientation system shown inFIG. 3 including a seed holder, in accordance with various embodimentsof the present disclosure.

FIG. 6 is an enlarged perspective view of the seed holder shown in FIG.5, in accordance with various embodiments of the present disclosure.

FIG. 7 is an enlarged side elevation view of the seed holder shown inFIG. 6, in accordance with various embodiments of the presentdisclosure.

FIG. 8 is a perspective view of a milling station and a seed transportsubsystem of the seed sampler system shown in FIG. 1, in accordance withvarious embodiments of the present disclosure.

FIG. 9 is a perspective view of a sampling station of the seed samplersystem shown in FIG. 1, in accordance with various embodiments of thepresent disclosure.

FIG. 10 is an enlarged side elevation view of the seed sampling station,shown in FIG. 9, during operation of the seed sampler system shown inFIG. 1, in accordance with various embodiments of the presentdisclosure.

FIG. 11 is a side elevation view of a liquid delivery apparatus of theseed sampling system, shown in FIG. 1, in a retracted position, inaccordance with various embodiments of the present disclosure.

FIG. 12 is a side elevation view of the liquid delivery apparatus shownin FIG. 11, in an extended position, in accordance with variousembodiments of the present disclosure.

FIG. 13 is a perspective view of a sample tray platform of the seedsampler system shown in FIG. 1, in accordance with various embodimentsof the present disclosure.

FIG. 14 is a perspective view of a seed treatment station of the seedsampler system shown in FIG. 1, in accordance with various embodimentsof the present disclosure.

FIG. 15 is a side elevation view of a seed conveyor of the seed samplersystem shown in FIG. 1, in accordance with various embodiments of thepresent disclosure.

FIG. 16 is a perspective view of a seed tray platform of the seedsampler system shown in FIG. 1, in accordance with various embodimentsof the present disclosure.

FIG. 17 is a side elevation view of a collection tube loading station ofthe seed sampler system shown in FIG. 1, in accordance with variousembodiments of the present disclosure.

FIG. 18 is a perspective view of a collection tube preparation subsystemof the seed sampler system shown in FIG. 1, in accordance with variousembodiments of the present disclosure.

FIG. 19 is a perspective view of a cleaning station of the seed samplersystem shown in FIG. 1, in accordance with various embodiments of thepresent disclosure.

Corresponding reference numerals indicate corresponding parts throughoutthe several views of the drawings.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is in no wayintended to limit the present teachings, application, or uses.Throughout this specification, like reference numerals will be used torefer to like elements.

FIG. 1 illustrates an automated seed sampler system 10, in accordancewith various embodiments of the present disclosure. Generally, the seedsampler system 10 includes a seed loading station 100, a seedorientation system 200, a seed transport subsystem 300, a millingstation 400, a sampling station 500, a sample collection and transportsubsystem 600, a liquid delivery subsystem 700, a sample depositsubsystem 800, a seed treatment station 900 and a seed deposit subsystem1000.

The seed sampler system 10 is structured and operable to isolate a seedfrom a seed bin 104 of the seed loading station 100, orient the seed atthe seed orientation station 200 and transfer the seed to the millingstation 400, via the transport subsystem 300. The seed sampler system 10is further structured and operable to remove a portion of the seed coatmaterial at the milling station 400, transfer the seed to the samplingstation 500, via the seed transport subsystem 300, where sample materialis extracted from the seed at the point where the seed coat material hasbeen removed. The seed sampler system 10 is still further structured andoperable to convey the extracted sample to the sample deposit subsystem800, via the sample transport subsystem 700, and deposit the extractedsample into a sample tray 14 located on the sample deposit subsystem800. In various embodiments, the sample material is collected in adisposable sample tube and delivered to the sample tray 14 using liquid,as described further below. Further yet, the seed sampler system 10 isstructured and operable to treat, e.g., apply a protective coating to,the exposed portion of the seed at the seed treatment station 900 andconvey the seed to the seed deposit subsystem 1000, where the seed isdeposited into a seed tray 18 located on a platform of the seed depositsubsystem 1000.

It should be understood that the seed sampler system 10, as shown anddescribed herein, includes various stationary braces, beams, platforms,pedestals, stands, etc. to which various components, devices,mechanisms, systems, subsystems, assemblies and sub-assemblies describedherein are coupled, connected and/or mounted. Although such braces,beams, platforms, pedestals, stands, etc. are necessary to theconstruction of the seed sampler system 10, description of theirplacement, orientation and interconnections are not necessary for oneskilled in the art to easily and fully comprehend the structure,function and operation of the seed sampler system 10. Particularly, suchbraces, beams, platforms, pedestals, stands, etc. are clearlyillustrated throughout the figures and, as such, their placement,orientation and interconnections are easily understood by one skilled inthe art. Therefore, for simplicity, such braces, beams, platforms,pedestals, stands, etc. will be referred to herein merely as systemsupport structures, absent further description of their placement,orientation and interconnections.

Referring now to FIGS. 2 and 3, in various embodiments, the seed loadingstation includes the seed bin 104 and a separating wheel 108. Theseparating wheel 108 is mounted for rotation in a vertical plane suchthat a portion of the separating wheel 108 extends into an interiorreservoir of the seed bin 104. Another portion of the separating wheel108 extends outside of the seed bin 104 such that a face 120 of theseparating wheel 108 is positioned adjacent a seed collector 124. Theseed separating wheel 108 includes a plurality of spaced apart recessedports 128 that extend through the face 120 and are communicativelycoupled to a vacuum system (not shown) such that a vacuum can beprovided at each of the recessed ports 128.

To initiate operation of the seed sampler system 10, seeds to be sampledand tested are placed in the seed bin 104 interior reservoir and avacuum is provided to at least some of the recessed ports 128, e.g., therecessed ports 128 in the face 120 of the portion of the separatingwheel 108 extending into the interior reservoir of the seed bin 104. Theseed separating wheel 108 is then incrementally rotated, via an indexingmotor 132, such that recessed ports 128 sequentially rotate through theinterior reservoir of the seed bin 104, out of the seed bin 104, andpast seed collector 124 before re-entering the interior reservoir of theseed bin 104. As the separating wheel incrementally rotates and therecessed ports 128 incrementally pass through the seed bin 104 interiorreservoir, individual seeds are picked up and held at each recessed port128 by the vacuum provided at the respective recessed ports 128. As theseparating wheel 108 incrementally rotates, the seeds are carried out ofthe seed bin 104 to the seed collector 124 where each seed is removedfrom the face 120 of the separating wheel 108. After each seed isremoved from the separating wheel 108, the seed is funneled to a loadingstation transfer tube 136. The seed is then passed through the loadingstation transfer tube 136, via gravity, vacuum or forced air, into aseed imaging fixture 204 of the seed orientation system 200. The loadingstation transfer tube 136 is sized to have an inside diameter that willonly allow the seed to pass through the loading station transfer tube136 in a longitudinal orientation. That is, the seed can only passthrough the loading station transfer tube 136 in either a tip-up ortip-down orientation and the inside diameter will not allow the seed totumble or flip as it passes through the loading station transfer tube136.

In various embodiments, the seed collector 124 includes a wiper (notshown) that physically dislodges each seed from the respective recessedport 128 as the separating wheel 108 incrementally rotates past the seedcollector 124. Thereafter, the dislodged seed passes through the loadingstation transfer tube 136 to the imaging fixture 204. Alternatively, invarious other embodiments, each seed can be released from respectiverecessed port 128 by temporarily terminating the vacuum at eachindividual recessed port 128 as the individual recessed port 128 ispositioned adjacent the seed collector 124. Thereafter, the dislodgedseed is transferred to the imaging fixture 204, via the loading stationtransfer tube 136. In still other embodiments, each seed can be blownfrom the respective recessed port 128 by temporarily providing forcedair at each individual recessed port 128 as the individual recessed port128 is positioned adjacent the seed collector 124. Thereafter, thedislodged seed is transferred to the imaging fixture 204, via theloading station transfer tube 136.

Additionally, in various embodiments the seed loading station 100 caninclude a bulk seed hopper 140 having a shaped surface and a vibratingfeeder mechanism 144. Large amounts of seed can be placed in the hopper140 where the seed is funneled onto the vibrating feed mechanism 144.The vibrating feeder mechanism 144 can be controlled to meter seeds intothe seed bin 104 where the seeds are separated and transferred to theimaging fixture 204 of the seed orienting system 200, as describedabove.

Referring now to FIGS. 3 and 4, the seed orientation system 200comprises the seed imaging fixture 204, an imaging device 208, and aseed orienting device 212 mounted to a stationary center platform 214 ofthe seed sampler system 10. The seed imaging fixture 204 includes awindow 216 and an internal seed orientation area that is visible throughthe window 216. The orienting device 212 includes a flipper actuator 220operable to rotate the seed while the seed is suspended in the seedorientation area. The imaging fixture 204 is connected to an end of theloading station transfer tube 136 and the imaging device 208 is mountedto a system support structure adjacent the imaging fixture such that theimaging device 208 is positioned to view a seed suspended in the seedorientation area through the window 216.

When a seed is transferred to the imaging fixture 204, via the loadingstation transfer tube 136, the seed is suspended within the seedorientation area, adjacent the window 216, and viewed by the imagingdevice 208 through the window 216. In various other embodiments, theseed is levitated within the seed orientation area using air providedthrough an orientation system transfer tube 224 connected to the bottomof the imaging fixture 204, opposite the loading station transfer tube136. Or, in various embodiments, the seed can be physically held withinthe seed orientation area using any suitable mechanical holding means.

As the seed is suspended adjacent the window 216, an image of the seedwithin the imaging fixture 204 is collected by the imaging device 208.The imaging device 208 can be any imaging device suitable for collectingimages through the window 216 of the seeds suspended within the seedorientation area. For example, in various embodiments, the imagingdevice 208 comprises a high speed, high resolution digital camera, suchas a disruptive visual technology (DVT) machine vision camera. The imageis communicated to a computer based system controller (not shown), wherean orientation of the seed, i.e., tip-up or tip-down, is determined. Ina various embodiments, the seed imaging device 208 additionally locatesa centroid of the seed and identifies the farthest point from thecentroid as the tip.

If the seed is determined to be tip-down, the seed is conveyed in thetip-down orientation, via the orientation system transfer tube 224, toone of a plurality of seed holders 304. If the seed is determined to betip-up, the flipper actuator 220 is commanded by the system controllerto rotate the seed 180° to place the seed in the tip-down orientation.For example, the flipper actuator 220 can be air-operated such that airis used to rotate the seed until the tip-down orientation is detected bythe imaging device 208. Or, the flipper actuator can be a mechanicalactuator that rotates the seed held by a suitable mechanical holdingdevice to place the seed in the tip-down orientation. Once in thetip-down orientation, the seed is conveyed in the tip-down orientation,via the orientation system transfer tube 224, to one of the seed holders304. Orienting the seeds in the tip-down position minimizes the impactto the seed's viability when a sample is removed from the seed, asdescribed below. In various embodiments, the seeds are conveyed via theorientation system transfer tube 224 utilizing gravity, i.e., the seedsfall from the imaging fixture 204, through the transfer tube 224 andinto one of the seed holders 304. Additionally, each seed is maintainedin the proper orientation, i.e., tip-down, during conveyance to therespective seed holder 304 by providing the orientation system transfertube 224 with an inside diameter sized such that the seeds cannot rotateto the tip-up position.

As used herein, the system controller can be a single computer basedsystem, or a plurality of subsystems networked together to coordinatethe simultaneous operations of the seed sample system 10, describedherein. For example, the system controller can include a plurality ofcontroller subsystems, e.g., a controller subsystem for each stationdescribed herein. Each controller subsystem could include one or moreprocessors or microprocessors that communicate with various seed samplersystem sensors, devices, mechanisms, motors, tools, etc., and arenetworked together with a main computer system to cooperatively operateall the stations, systems and subsystems of the seed sampler system 10.Or alternatively, the system controller could comprise a single computercommunicatively connected to all the various sensors, devices,mechanisms, motors, tools, etc., to cooperatively operate all thestations, systems and subsystems of the seed sampler system 10.

The seed holders 304 are mounted to, and equally spaced around aperimeter area of, a motorized turntable 308 of the seed transportsubsystem 300. The orientation system transfer tube 224 is connected ata first end to the seed imaging fixture 204 such that a second end ofthe orientation system transfer tube 224 is positioned a specificdistance above a perimeter portion of the turntable 308. Moreparticularly, the second end of the orientation system transfer tube 224is positioned above the turntable 308 a distance sufficient to allow theseed holders 304 to pass under the orientation system transfer tubesecond end. However, the second end of the orientation system transfertube 224 is also positioned above the turntable 308 such that there isonly a small amount of clearance between the second end and the holders304. Therefore, each seed will remain in the tip-down orientation as ittransitions from the orientation system transfer tube 224 to one of theseed holders 304.

Referring now to FIGS. 5, 6 and 7, each seed holder 304 is structuredand used to rigidly retain a respective seed in the tip-downorientation. Each seed holder 304 includes a pair of opposing clampheads 312 slidingly positioned within opposing clamp pockets 316. Theopposing clamp pockets 316 are separated by a seed channel 318 laterallyformed along a centerline C of the seed holder 304. Each clamp head 312is connected to a respective clamp piston 320 via a respective clampshaft 324. Each clamp piston 320 is slidingly housed within a respectivelongitudinal internal piston cylinder 328 of seed holder 304. Acompression spring 332 is positioned within each piston cylinder 328between a base of the respective piston and a bottom of the respectivepiston cylinder 328. Accordingly, each clamp head 312 is biased towardthe centerline C of the seed holder 304. When a seed holder 304 is in anidle state, that is, when the respective seed holder is not holding aseed or being manipulated to hold a seed, the opposing clamp heads 312will be biased by the springs 332 to a fully extended, or deployed,position. When the clamp heads 312 are in the deployed position, a topof each respective piston 320 will extend into a respective forkpassageway 336 extending laterally through the seed holder 304 onopposing sides of the seed channel 318.

Each clamp head 312 is fabricated from a slightly soft, resilientmaterial, such as neoprene, such that a seed held between the opposingclamp heads 312, as described below, will not be damaged.

As described above, the seed holders 304 are mounted to, and equallyspaced around a perimeter area of, the turntable 308. Prior to,subsequent to, or substantially simultaneously with the seed orientationprocess described above, the turntable 308 is rotated to place an empty,i.e., absent a seed, seed holder 308 under the orientation systemtransfer tube 224. More specifically, the seed channel 318 is positionedunder the orientation system transfer tube 224. When a seed holder 304is positioned under the orientation system transfer tube 224 anautomated clamp head spreader 340 is activated to spread the clamp heads312 such that a seed can be received between the clamp heads 312. Theclamp head spreader 340 is mounted to system support structure adjacentthe seed orienting device 212 and includes a pair of fork tangs 344coupled to a fork base 348. The clamp head spreader 340 is operable toextend the fork base 348 and tangs 344 toward the seed holder 304. Forexample, the clamp head spreader 340 can be a pneumatic device operableto extend and retract the fork base 348. Each fork tang 344 has achamfered distal end portion and is sized to fit within the forkpassageways 336.

Upon activation of the clamp head spreader 340, the fork base 348 isextended toward the seed holder 304 such that the tangs 344 are insertedinto the fork passageways 336. As each tang 344 slides into therespective fork passageway 336 the chamfered distal end portions slidebetween the top of each respective piston 320 and an inner wall of thefork passageway 336. As the tangs 344 are extended further into eachfork passageway 336, the chamfer of each tang forces the respectivepiston 320 outward and away from the centerline C of the seed holder.Accordingly, as the pistons 320 are moved outward and away from thecenterline C, the clamp heads 312 are also moved outward and away fromeach other and the centerline C. Thus, the clamp heads 312 are moved toa retracted position where a seed can be placed between them.

Once the clamp heads 312 have been retracted, a properly oriented seedcan be conveyed through the orientation system transfer tube 224 andpositioned in the tip-down orientation between the clamp heads 312. Invarious embodiments, the seed sampler system 10 additionally includes aseed height positioning subsystem 360 for positioning the seed at aspecific height within the respective seed holder 304. The seed heightpositioning subsystem includes a vertical positioner 364 mounted tosystem support structure below the perimeter area of the turntable 308,directly opposite the orientation system transfer tube 224, and a datumplate actuator 368 mounted to the center platform 214 directly oppositethe clamp head spreader 340. The vertical positioner 364 includes aspring loaded plunger 372 mounted to a positioner head 376 and the datumplate actuator 368 includes a datum plate 380 mounted to a datum plateactuator head 384. The vertical positioner 364 is operable to extend thepositioner head 376 and plunger 372 toward a bottom of the turntable 308directly opposite the seed holder centerline C. For example, thevertical positioner 364 can be a pneumatic device operable to extend andretract the plunger 372. Similarly, the datum plate actuator 368 isoperable to extend the actuator head 384 and datum plate 380 over thetop of the seed holder seed channel 318. For example, the datum plateactuator 368 can be a pneumatic device operable to extend and retractthe datum plate 380.

Once the seed has been positioned between the retracted clamp heads 312,the positioner head 376 is extended upward to insert a plunger shaft 388through a hole (not shown) in the bottom of the turntable 308 and acoaxially aligned hole (not shown) in the bottom of the seed holder seedchannel 318. Substantially simultaneously, the datum plate actuator 368extends the actuator head 384 to position the datum plate 380 aspecified distance above the seed holder 304, directly above the hole inthe bottom of the seed holder seed channel 318. More specifically, aspositioner head 376 is moved upward, the plunger shaft 388 is extendedinto the coaxially aligned holes and contacts the tip of the seed. Theseed is then pushed upward between the clamp heads 312 until the crownof the seed contacts the datum plate 380. The spring loaded structure ofthe plunger 372 allows the shaft 388 to retract within the plunger 372when the seed crown contacts the datum plate 380 so that the seed isheld in place without damaging the seed. Accordingly, the crown of theseed is located at a specific height relative to the top of theturntable 308.

With the seed crown held against the datum plate 380 by the springloaded plunger 372, the clamp head spreader 340 is operated to retractthe fork base 348 and withdraw the tangs 344 from the respectivepassageways 336. Upon withdrawal of the tangs 344, the springs 332 biasthe clamp heads 312 toward the deployed position and firmly clamp theseed between the clamp heads 312. The datum plate 380 and plunger shaft388 are subsequently retracted leaving the seed properly positioned, or‘loaded’, in the respective seed holder 304. The system controller thenrotates the turntable 308 to position the ‘loaded’ seed holder 304beneath the milling station 400 and the next empty seed holder 304beneath the seed orienting device 212.

Referring now to FIG. 8, as described above, the seed sampler system 10includes the seed transport subsystem 300 for conveying the seedsbetween individual stations of the sampler system, e.g., the seedloading station 100, milling station 400, sampling station 500, etc.Generally, the seed transport subsystem 300 can be any suitableconveyance mechanism such as, for example, a belt conveyor, rollerconveyor, and the like. In various embodiments, however, the transportsubsystem 300 comprises the round turntable 308 that is pivotallymounted at its center for rotation. The turntable 308 is virtuallydivided into a plurality of sectors, with each sector containing a seedholder 304. The number of sectors available on the turntable 308 may beeven or odd with a number chosen which depends in large part on thediameter of the turntable 308, the size of the seed holders 304 and theneeds of the transport application.

The circular turntable 308 is pivotally mounted at its center to a shaftand bearing system 390. In various embodiments, a shaft (not shown) ofthe shaft and bearing system 390 can be directly coupled to an actuatingmotor 392. Alternatively, the shaft may be separate from the actuatingmotor 392 and driven for rotation by a suitable chain drive, pulleydrive or gear drive. In various implementations, the actuating motor 392can be a high torque stepper motor.

In operation, the actuating motor 392 for the turntable 308 is actuatedto step forward (which can be either clockwise or counter clockwise,depending on configuration) to rotationally move the turntable 308 fromstation to station of the sampler system 10. Therefore, the seed holders304 are aligned with auxiliary devices, such as the loading station 100,milling station 400, sampling station 500, etc. In this configuration,an auxiliary device can be positioned about the turntable 308 atstations which are in alignment with each position and thus have preciseaccess to the seeds and seed holders 304. To the extent necessary, theperipheral edges of the turntable 308 may be supported with rollers,guides, slides, or the like, to assist with smooth rotation of theturntable conveyor.

Referring to FIG. 8 further, as described above, once each seed holder304 is ‘loaded’ with a seed, the system controller rotates the turntable308 to position the ‘loaded’ seed holder 304 beneath the milling station400. The milling station 400 includes at least one milling tool 404mounted to system support structure above the perimeter area of theturntable 308. The one or more milling tools 404 are used to remove aportion of the seed coat from each seed when the respective seed holder304 is positioned beneath the milling station 400. Each milling tool 404includes a Z-axis actuator 408 operable to lower and raise at least aportion of the respective milling tool 404 along the Z-axis. Eachmilling tool 404 is controlled by the system controller and can beelectrically, pneumatically or hydraulically operated.

The milling tool(s) 404 can be any suitable mechanism for removing aportion of seed coat material from each seed. For example, in variousembodiments, each milling tool 404 is a rotary device including theZ-axis actuator 408 and a rotary drive 412 operationally coupled to abit chuck 416. Each Z-axis actuator 408 is operable to lower and raisethe respective bit chuck 416 and a milling tool bit 420 held within thebit chuck 416 along the Z-axis. The milling tool bit 420 can be anyinstrument suitable for removing the seed coat material, such as a millbit, drill bit, a router bit, a broach, or a scraping tool. For example,in various embodiments, the milling tool bit 420 comprises an end millbit. Each Z-axis actuator 408 is controlled by the system controller tolower the respective Z-axis actuator 408 a specific predetermineddistance. The rotary drive 412 of each rotary milling tool 404 functionsto rotate, or spin, the respective bit chuck 416 and any milling toolbit 420 held within the bit chuck 416.

In operation, when a seed holder 304 is positioned below a rotarymilling tool 404, the rotary drive 412 is activated to begin spinningthe bit chuck 416 and milling tool bit 420. The Z-axis actuator 408 isthen commanded to lower the respective bit chuck 416 and milling toolbit 420 a specific predetermined distance. As the spinning milling toolbit 420 is lowered, it contacts the crown of the seed and removes theseed coat from at least a portion of the crown. This exposes a portionof the inner seed material that can be extracted and utilized to testand analyze the various traits of the respective seed, as describedbelow.

In various embodiments, the milling station 400 comprises at least twomilling tools 404 mounted to a milling station horizontal movement stage424 that is mounted to system support structure. The milling stationhorizontal movement stage 424 is controlled by the system controller toposition a selected one of the milling tools 404 above a seed holder 304positioned below the milling station 400. The selected milling tool 404is then operated as described above to remove the seed coat from atleast a portion of the respective seed crown. Subsequently, the systemcontroller can position a second one of the milling tools 404 above asubsequent seed holder 304 positioned below the milling station 400. Thesecond selected milling tool 404 is then operated as described above toremove the seed coat from at least a portion of the respective seedcrown. In such embodiments, the milling station 400 can additionallyinclude at least one milling bit cleaning assembly 428 for cleaning thebit 416 of the idle, i.e., not in use, milling tool 404. That is, whileone milling tool 404 is operable to remove the seed coat from arespective seed, the bit 420 of an idle second milling tool 404 can becleaned by a cleaning assembly 428 in preparation for the next millingoperation. In various embodiments, the milling bit cleaning assemblies428 utilize air pressure and or vacuum pressure to remove and/or collectany seed coat residue that may collect on the bits 420 of the millingtools 404.

Referring now to FIG. 9, once the seed coat has been removed from aseed, the system controller rotates the turntable 308 to position therespective seed holder 304 beneath the sampling station 500. Thesampling station 500 includes at least one sampling tool 504 mounted tosystem support structure anchored to the center platform 214 above theturntable 308. The one or more sampling tools 504 are used to remove aportion, i.e., a sample, of the exposed inner seed material when therespective seed holder 304 is positioned beneath the sampling station500. Each sampling tool 504 includes a Z-axis actuator 508 operable tolower and raise at least a portion of the respective sampling tool 504along the Z-axis. Each sampling tool 504 is controlled by the systemcontroller and can be electrically, pneumatically or hydraulicallyoperated.

The sampling tool(s) 504 can be any suitable mechanism for removing asample of the exposed inner seed material from each seed. For example,in various embodiments, each sampling tool 504 is a rotary deviceincluding the Z-axis actuator 508 and a rotary drive 512 operationallycoupled to a bit chuck 516. Each Z-axis actuator 508 is operable tolower and raise the respective bit chuck 516 and a sampling tool bit 520held within the bit chuck 516 along the Z-axis. The sampling tool bit520 can be any instrument having an outer diameter smaller than thecircumference of the area of exposed inner seed material, and suitablefor removing a sample from the exposed inner seed material, such as adrill bit, a router bit, a broach, or a coring tube. It is importantthat the sampling tool bit 520 be of a smaller diameter than the millingtool bit 420 to ensure that sample material is obtained from an areawhere the seed coat material has been removed, thereby substantiallyeliminating any seed coat material from contaminating the samplematerial collected.

For example, in various embodiments, the sampling tool bit 520 comprisesa spade tip drill bit having an outer diameter that is smaller than anouter diameter of the milling tool bit 420. Each Z-axis actuator 508 iscontrolled by the system controller to lower the respective Z-axisactuator 508 a specific predetermined distance. The rotary drive 512 ofeach rotary sampling tool 454 functions to rotate, or spin, therespective bit chuck 516 and any sampling tool bit 520 held within thebit chuck 516.

In operation, when a seed holder 304 is positioned below a rotarysampling tool 504, the rotary drive 512 is activated to begin spinningthe bit chuck 516 and sampling tool bit 520. The Z-axis actuator 508 isthen commanded to lower the respective bit chuck 516 and sampling toolbit 520 a specific predetermined distance. As the spinning sampling toolbit 520 is lowered, it contacts the exposed inner material of the seedand cuts away a sample of the inner material. The sample is thenremoved, or extracted, to be tested and analyzed for various traitsand/or characteristics of the respective seed, as described below.

In various embodiments, the sampling station 500 comprises at least twosampling tools 504 mounted to a sampling station horizontal movementstage 524 that is mounted to system support structure. The samplingstation horizontal movement stage 524 is controlled by the systemcontroller to position a selected one of the sampling tools 504 above aseed holder 304 positioned below the sampling station 500. The selectedsampling tool 504 is then operated as described above to remove thesample from the exposed inner material of the respective seed.Subsequently, the system controller can position a second one of thesampling tools 504 above a subsequent seed holder 304 positioned belowthe sampling station 500. The second selected sampling tool 504 is thenoperated as described above to remove the sample from the exposed innermaterial of the respective seed. In such embodiments, the samplingstation 500 can additionally include at least one sampling bit cleaningassembly 528 for cleaning the sampling bit 520 of the idle, i.e., not inuse, sampling tool 504. That is, while one sampling tool 504 is operableto remove the sample from a respective seed, the sampling bit 520 of anidle second sampling tool 504 can be cleaned by a sampling bit cleaningassembly 528 in preparation for the next sampling operation. In variousembodiments, the sampling bit cleaning assemblies 528 utilize airpressure and or vacuum pressure to remove and/or collect any inner seedmaterial residue that may collect on the sampling bits 520 of thesampling tools 504.

Referring now to FIGS. 9 and 10, the sample collection and transport(SCT) subsystem 600 is controlled by the system controller to operate insynchronized coordination with the sampling station 500 to collect eachsample as it is removed from each seed. The SCT subsystem 600 includes amotorized rotating platform 604 driven by an actuating motor (not shown)similar to the turntable 308 actuating motor 392 (shown in FIG. 8). TheSCT subsystem additionally includes a plurality of collection tubeplacement (CTP) devices 608 equally spaced around, and mounted to aperimeter area of the rotating platform 604. Each CTP device 608includes a pivot bar 612 having a hollow tube mount 616 mounted througha transverse bore (not shown) in the pivot bar 612. The tube mount 616includes a distal end 618 structured to accept a base 620 of acollection tube 624 and a proximal end 628 adapted to receive pneumatictubing (not shown).

Each CTP device 608 further includes a pivot bar actuator 632controllable by the system controller to rotate the pivot bar 612 tovarious positions about a longitudinal axis of the pivot bar 612. Invarious embodiments, the pivot bar actuator 632 is operable to pivot thetube mount 616 between a flushing position, as illustrated in FIG. 11, acollection position, as illustrated in FIG. 10, and a load and depositposition, as illustrated in FIGS. 13 and 17. The CTP device 608additionally includes a stop arm 636 connected to the pivot bar 612 andan adjustable stop 640, e.g., a set screw, adjustably engaged with thestop arm 636. The stop arm 636 and adjustable stop 640 pivot with thepivot bar 612 and function to accurately stop rotation of the pivot bar612 so that the tube mount 616 is in the collection position.

Simultaneously with the operation of the seed loading station 100, themilling station 400 and the sampling station 500, the SCT subsystem 600operates to load the collection tube 624 on the tube mounts 616 of eachCTP device 608, collect the samples in the collection tubes 624 as eachsample is being removed, and deposit the collected samples in the sampletrays 14. Loading the collection tubes 624 on the tube mounts 616 anddepositing the collected sample in the sample trays 14, will bedescribed further below with reference to FIG. 17, and FIGS. 12 and 13,respectively. The collection tubes 624 can be any container or devicesuitable for mounting on the tube mounts 616 and collecting the samplesas described below. For example, in various embodiments, the collectiontubes 624 are disposable such that each sample is collected in a cleancollection tube 624. An example of such a disposable collection tube 624is a filtered pipette.

As described above, the SCT subsystem 600 is controlled by the systemcontroller to operate in synchronized coordination with the samplingstation 500 to collect each sample as it is removed from each seed. Morespecifically, prior to removing the sample from the seed, the systemcontroller rotates the platform 604 to position a CTP device 608adjacent the sampling station 500. Particularly, a CTP device 608 ispositioned adjacent the sampling station 500 such that the respectivetube mount 616 is aligned with the seed held within an adjacent seedholder 304 that has been positioned below a sampling device 504, via thecontrolled rotation of the turntable 308. Prior to positioning the CTPdevice 608 adjacent the seed holder 304 positioned at the samplingstation 500, the SCT system 600 has loaded a collection tube 624 on therespective tube mount distal end 618 and the respective pivot baractuator 632 has raised the collection tube 624 to a position above thecollection position, e.g., the flushing position. Once the CTP device608 is positioned adjacent the respective seed holder 304, the pivot baractuator 632 lowers the loaded collection tube 624 until the adjustablestop 640 contacts a stop plate 648 mounted to system support structurebetween the turntable 308 and the platform 604 adjacent the samplingstation 500. The adjustable stop 640 is preset, i.e., pre-adjusted, suchthat the rotation of the pivot bar 612 is stopped to precisely locate atip 672 of the collection tube 624 in very close proximity to, or incontact with, the crown of the seed held in the adjacent seed holder304.

The sampling bit 620 of a sampling tool 504 is then lowered to beginremoving the sample, as described above. As sampling bit 620 is lowered,a vacuum is provided at the collection tube tip 672. The vacuum isprovided via vacuum tube (not shown) connected to the proximal end 628of the tube mount 616. The vacuum tube is also connected to a vacuumsource (not shown) such that the vacuum is through the vacuum tube, thehollow tube mount 616 and the collection tube 624. Accordingly, as thesampling bit 620 removes the sample material, the sample is drawn intothe collection tube 624, where the sample is collected. In variousembodiments, the sampling station 500 can include a positive pressuredevice (not shown) to assist the vacuum provided at the respective seedto collect substantially all the removed sample in the respectivecollection tube 624.

Each collection tube includes a filter 676 that prevents the sample frombeing drawn into the tube mount 616 and vacuum tube. Once the sample hasbeen collected, the pivot bar actuator 632 raises the collection tube624 to the flush position and the respective CTP device 608 is advancedto a position adjacent the liquid delivery subsystem 700. Consequently,another CTP device 608 and empty collection tube 624 are positionedadjacent a subsequent seed holder 304 and un-sampled seed that have beenmoved to the sampling station.

Referring now to FIGS. 11 and 12, the liquid delivery subsystem 700includes a liquid injection device 704 mounted to a linear actuator 708operable to extend and retract the liquid injection device 704 along alinear axis M. More specifically, the linear actuator 708 is operable toinsert and withdraw an injection needle 712, fastened to the liquidinjection device 704, into and out of the tip 672 of the respectivecollection tube 624. When a collection tube 624 with a collected samplehas been raised to the flush position and advanced to be positionedadjacent the liquid delivery subsystem 700, the linear actuator 708 andinjection needle 712 are in the retracted position, as illustrated inFIG. 11. The pivot bar actuator 632 and rotating platform 604 arecontrolled by the system controller such that when the CTP device 608 isadjacent the liquid delivery subsystem 700 and the collection tube 624is raised to the flush position, a linear axis of the collection tube624 is substantially coaxial with the linear axis M of the liquidinjection device 704, as shown in FIG. 11.

Once the linear axis of the collection tube 624 is positioned to becoaxial with the M axis, the linear actuator 708 extends to insert theinjection needle 712 into the tip 672 of the collection tube 624. Theliquid injection device 704 is connected to an extraction fluid supplysource (not shown) via a fluid port 716 coupled to a metering valve 720of the liquid injection device 704. Therefore, once the injection needle712 is inserted into the collection tube tip 672, the fluid injectiondevice 704 injects a metered amount of extraction fluid into thecollection tube 624. The injected extraction fluid flushes, or washes,the interior sides of the collection tube 624 and creates an aqueoussolution with the respective sample, herein referred to as an aqueoussample. Thus, any of the collected sample that may have gathered on theinterior walls of the collection tube 624 is flushed off so thatsubstantially all the collected sample is suspended in the resultingaqueous solution. The extraction liquid can be any liquid suitable fordelivering substantially all the sample material collected within eachrespective collection tube 624, without interfering with the desiredanalysis, e.g., chemical and genetic analysis, of the sample material.For example, in various embodiments, the extraction liquid may comprisedistilled water or any suitable solvent compatible with the desiredsample analysis.

Once the collected sample has been mixed with the extraction liquid, thelinear actuator 708 retracts to withdraw the injection needle 712 fromcollection tube tip 672. The system controller then advances therotating platform 604 to position the CTP device 608 above the sampledeposit subsystem 800. The system controller additionally commands therespective pivot bar actuator 632 to position the collection tube in theload and deposit position. The load and deposit position points the tubemount 616 and mounted collection tube 624 downward to a substantiallyvertical orientation.

Referring now to FIG. 13, the sample deposit subsystem 800 includes asample tray platform 804 adapted to securely retain a plurality ofsample trays 14 in fixed positions and orientations. Each sample tray 14includes a plurality of sample wells 22, each of which are adapted forreceiving a collected aqueous sample. The sample tray platform 804 ismounted to an X-Y stage 808. The X-Y stage 808 is a two-dimensionaltranslation mechanism, including a first translating track 812 and asecond translating track 816. The X-Y stage 808 additionally includes afirst linear actuator 818 operable to bidirectionally move a firstcarriage (not shown) along the length of the first translating track812. The X-Y stage 808 further includes a second linear actuator 820operable to bidirectionally move a second carriage (not shown) along thelength of the second translating track 816. The second translating track816 is mounted to the first carriage and the sample tray platform 804 ismounted to the second carriage.

The first and second linear actuators 818 and 820 are controlled by thesystem controller to precisely move the sample tray platform 804 in twodimensions. More particularly, the first and second actuators 818 and820 move the sample tray platform 804 within an X-Y coordinate system toprecisely position any selected well 22 of any selected sample tray 14at a target location beneath the CTP device 608 holding the collectiontube 624 containing the collected aqueous sample. The target location isthe location in the X-Y coordinate system that is directly below thecollection tube tip 672 when the collection tube 624 is in the load anddeposit position above the sample tray platform 804. Thus, once the CTPdevice 608 is positioned above the sample tray platform 804 and therespective collection tube 624 is placed in the load and depositposition, with the tip 672 pointing at the target location, the systemcontroller positions a selected well 22, of a selected sample tray 14 atthe target location. The aqueous sample is then deposited into theselected well 22 by providing positive pressure to the proximal end 628of tube mount 616.

As the sample trays 14 are placed on the sample tray platform 804, atray identification number, e.g., a bar code, for each sample tray 14and the location of each sample tray 14 on the platform 804 is recorded.Additionally, as each aqueous solution is deposited in a well 22, an X-Ylocation of the well, i.e., the target location, on the sample trayplatform 804 can be recorded. The recorded tray and well positions onthe sample tray platform 804 can then be compared to the X-Y locationsof each deposited aqueous sample, to identify the specific aqueoussample in each well 22 of each sample tray 14.

Once each aqueous sample is deposited into a selected well 22, thesystem controller advances the rotating platform 604 to position asubsequent CTP device 608, holding a collection tube 624 containing asubsequent aqueous sample, above the sample deposit subsystem 800.Additionally, the CTP device 608 holding the used, empty collection tube624 is advanced to a collection tube discard station 850 (shown inFIG. 1) where the used collection tube 624 can be removed or ejectedfrom the respective tube mount 616 and discarded. Referring briefly toFIG. 1, in various embodiments, the collection tube discard station 850includes a collection tube removal device 854 mounted to a linearactuator 858 operable to extend and retract an automated gripper 862.When a CTP device 608 holding a used collection tube 624 is positionedadjacent the collection tube removal device 854, the system controllercommands the linear actuator 858 to extend and gripper 862 to grasp theused collection tube 624. The system controller then commands the linearactuator 858 to retract, thereby removing the used collection tube 624from the respective tube mount 616. The gripper 862 can then becommanded to release the used collection tube 624 allowing it to fallinto a discard container (not shown).

Referring now to FIG. 14, in various embodiments, after a seed has had asample extracted at the sampling station 500, the system controller mayadvance the turntable 308 to position the respective seed holder 304adjacent a seed treatment station 900. The seed treatment station 900includes a treatment dispenser 904 mounted to system support structureabove the perimeter area of the turntable 308. The treatment dispenser904 includes an applicator 908 configured to apply a seed treatment suchas a sealant to the exposed portion of the respective seed, i.e., thearea of the seed crown where the seed coat has been removed and thesample extracted. The seed treatment can be any substance designed toenhance one or more properties of the seed or to protect the seed frombacteria or other harmful elements that could damage the seed anddestroy the germination viability of the seed. For example, in variousembodiments, the seed treatment is a sealant comprising a fungicideand/or polymer delivered to the seed by the treatment dispenser 904 viathe applicator 908. The applicator 908 can be any device suitable toapply the desired seed treatment to the seeds, for example, a brush,needle or nozzle. In various embodiments, the applicator 908 comprises aspray nozzle and the treatment dispenser 904 includes a fluid port 912coupled to a metering valve 916. In such embodiments, the treatmentdispenser 904 is connected to liquid seed treatment supply source (notshown) via the fluid port 912. Accordingly, when a seed holder 304 ispositioned at the seed treatment station 900, beneath the treatmentdispenser 904, the system controller commands the treatment dispenser904 spray a metered amount of seed treatment on the respective seed.

Referring now to FIGS. 15 and 16, after sampling and the optional seedtreatment, the system controller advances the turntable 308 until therespective seed holder 304 is positioned adjacent a second clamp headspreader 1004 of the seed deposit subsystem 1000. The clamp headspreader 1004 is mounted to system support structure and includes a pairof fork tangs 1008 coupled to a fork base 1012. The clamp head spreader1004 is substantially identical in form and function as the clamp headspreader 340 described above with reference to FIG. 5. Accordingly, uponactivation of the clamp head spreader 1004, the fork base 1012 isextended toward the seed holder 304 such that the tangs 1008 areinserted into the fork passageways 336. As the tangs 1008 slide into therespective fork passageways 336, the clamp heads 312 of the respectiveseed holder 304 are retracted, as similarly described above. As theclamp heads 312 retract, the respective seed is allowed to fall throughthe coaxially aligned holes in the bottom of the seed holder seedchannel 318 and the turntable 308 into a funnel 1016 of a seed conveyor1020.

The seed conveyor 1020 comprises a first tube section 1024 coupled at afirst end to the funnel 1016 and to an inlet of a first venturi device1028 at a second end. A second tube section 1032 is connected at a firstend to an outlet of the first venturi device 1028 and at a second end toan inlet of a second venturi device 1036. An outlet of the secondventuri device 1036 is connected to seed dispenser 1040 that is mountedto system support structure above a seed tray platform 1044. The firstventuri device 1028 is operable to induce an air flow in the first andsecond tube sections 1024 and 1032 toward the seed dispenser 1040. Atthe same time, the second venturi device 1036 is operable to induce anair flow toward the funnel 1016. Thus, the air flow induced by the firstventuri device 1028 will draw the seed into the first funnel 1016 andfirst tube section 1020. Additionally, as the seed enters the first tubesection 1024 it is propelled toward the seed dispenser 1040 by the airflow provided by the first venturi device 1028. Subsequently, as theseed nears the seed dispenser 1040, the seed is slowed down by the airflow provided by the second venturi device 1036 so that the seed isgently dispensed from the seed dispenser 1040, into a seed tray 18without damaging the seed. In various embodiments, the air flow providedby the second venturi 1036 actually stops the movement of the seed,allowing the seed to drop under gravity into a seed tray 18. Variousposition sensors (not shown) can be provided on the first and secondtube sections 1024 and 1032 to detect the presence of the seed, andprovide input to the system controller to control operation of the seedconveyor 1020.

Referring particularly to FIG. 16, the seed deposit subsystem 1000additionally includes a seed tray platform 1044 adapted to securelyretain a plurality of seed trays 18 in fixed positions and orientations.Each seed tray 18 includes a plurality of seed wells 26, each of whichare adapted for receiving a seed dispensed from the seed dispenser 1040.The seed dispenser 1040 is mounted to system support structure above theseed tray platform 1044 such that seeds can be dispensed from the seeddispenser 1040 into selected seed wells 26 of selected seed trays 18.

The seed tray platform 1044 is mounted to an X-Y stage 1048. The X-Ystage 1048 is a two-dimensional translation mechanism, including a firsttranslating track 1052 and a second translating track 1056. The X-Ystage 1048 additionally includes a first linear actuator 1060 operableto bidirectionally move a first carriage (not shown) along the length ofthe first translating track 1052. The X-Y stage 1048 further includes asecond linear actuator 1064 operable to bidirectionally move a secondcarriage (not shown) along the length of the second translating track1056. The second translating track 1056 is mounted to the first carriageand the seed tray platform 1044 is mounted to the second carriage.

The first and second linear actuators 1060 and 1064 are controlled bythe system controller to precisely move the seed tray platform 1044 intwo dimensions. More particularly, the first and second actuators 1060and 1064 move the seed tray platform 1044 within an X-Y coordinatesystem to precisely position any selected well 26 of any selected seedtray 18 at a target location beneath the seed dispenser 1040. The targetlocation is the location in the X-Y coordinate system that is directlybelow a tip 1068 of the seed dispenser 1040. Once a seed holder 304 ispositioned above the funnel 1016, the system controller positions aselected well 26, of a selected seed tray at the target location. Theseed in the seed holder 304 is released into the funnel 1016 andtransported to seed dispenser 1040, as described above, and gentlydeposited into the selected well.

As the seed trays 18 are placed on the seed tray platform 1044, a trayidentification number, e.g., a bar code, for each seed tray 18 and thelocation of each seed tray 18 on the seed tray platform 1044 isrecorded. Additionally, as each seed is deposited in a well 26, an X-Ylocation of the well, i.e., the target location, on the seed trayplatform 1044 can be recorded. The recorded tray and well positions onthe sample tray platform 1044 can then be compared to the X-Y locationsof each deposited seed, to identify the specific seed in each well 26 ofeach seed tray 18.

As described above, each of the seed trays 18 and the sample trays 14include a plurality of wells 26 and 22, respectively. In variousembodiments, the number and arrangement of the wells 26 in the seedtrays 18 corresponds to the number and arrangement of the wells 22 inthe sample trays 14. This facilitates a one-to-one correspondencebetween a seed and its extracted sample. However, in some embodiments,it may be desirable to provide multiple wells 22 in the sample trays 14for each well 26 in the seed trays 18, for example, where multiple testsmay be run on the samples, or where different samples may be taken fromthe same seed (e.g. samples from different depths).

Referring now to FIG. 17, in various embodiments, the seed samplersystem 10 additionally includes a collection tube loading station 1100for mounting the collection tubes 624 on the tube mounts 616 of each CTPdevice 608. The tube loading station 1100 includes a hopper 1104 havinga shaped surface and a vibrating feeder chute 1108 extending from anopen bottom of the hopper 1104. Large amounts of collection tubes 624can be deposited into the hopper 1104 where the vibrating feeder chute1108 feeds the collection tubes 624 into a vibrating bowl feeder 1112. Agravity based feed track 1116 is connected to an outlet 1118 of thevibrating bowl feeder 1112 at a first end 1116A. A second end of thefeed track 1116 terminates at a collection tube ram device 1120. The ramdevice 1120 extends orthogonally downward from the feed track second end1116B and includes a longitudinal lift channel 1124 extending along thelength of the ram device 1120. The ram device 1120 additionally includesa push mechanism (not shown) internal to the ram device 1120. The pushmechanism can be any mechanism operable to push a collection tube 624,longitudinally positioned within the lift channel 1124, out an upper end1120A of the ram device 1120. For example, the push mechanism caninclude a linear actuator that drives a ram shaped to receive at least aportion of a collection tube 624.

As the vibrating feeder bowl 1112 vibrates, collection tubes 624 migratetoward the outlet 1118 of the vibrating bowl feeder 1112. At the outlet1118, the collection tubes 624 fall into the feed track first end 1116Athat is shaped to cause the collection tubes 624 fall into a tube slot(not shown) that extends the length of the feed track 1116. Morespecifically, the collection tubes 624 are caused to fall tip-down intothe tube slot and hang within the tube slot by a lip 620A of thecollection tube base 620 (shown in FIG. 10). Gravity and vibration fromthe vibrating feeder bowl 1112 cause the collection tubes 624 to travelthe length of the feed track 1116 and accumulate, single-file, at thefeed track second end 1116B. As the collection tubes 624 accumulate,single-file at the second end 1116 the lead collection tube 624 will belongitudinally oriented within the longitudinal lift channel. The ramdevice 1120 is then actuated such that the push mechanism pushes thelead collection tube 624 out the upper end 1124A of the ram device liftchannel 1124.

Prior to actuating the ram device 1120, the system controller willadvance the rotating platform 604 to position a CTP device 608 above thesecond end 1116B of the feed track 1116. The system controller willfurther command the pivot bar actuator 632 to position the tube mount616 in the load and deposit position, such that the tube mount distalend 618 is directly above the lift channel upper end 1124A. Therefore,as the lead collection tube is pushed, or lifted, out of the liftchannel upper end 1124A the collection tube base 620 is pushed onto thetube mount distal end 618. The tube mount distal end 618 is sized suchthat there will be a friction fit between the collection tube base 620and the tube mount distal end 618. Accordingly, the collection tube 624is lifted out of the ram device 1120 and mounted on the respective tubemount. The next collection tube 624 in the feed track 1116 will then bepositioned within the lift channel 1124 and a subsequent tube mountdistal end 618 positioned to receive the collection tube 624.

Referring now to FIG. 18, in various embodiments, the collection tubes624 can comprise commercially available pipettes, referred to herein aspipettes 624′. In such embodiments, the pipettes 624′ may require aportion of the tip 672′ be removed to allow for proper extraction of thesample, flushing of the pipette, and depositing of the aqueous sample inthe sample trays 14. Therefore, in such embodiments, the seed samplersystem 10 can include a collection tube preparation subsystem 1150operable to cut off a portion of each pipette tip 672′ after eachpipette 624′ has been mounted on a respective tube mount 616. Thecollection tube preparation subsystem 1150 includes a linear actuator1154 operable to extend and retract a base 1158A of a cutter 1158 alonga linear axis P. The linear actuator 1154 is mounted to system supportstructure below the rotating platform 604 such that when a newly mountedpipette 624′, i.e., the pipette 624′ has just been mounted on therespective tube mount 616, is advanced to the collection tubepreparation subsystem 1150, the pipette tip 672′ is positioned within acutting chamber 1162.

The cutting chamber 1162 is formed between the cutter base 1158A and acutting recess 1166 formed in a head 1158B of the cutter 1158. Asillustrated in FIG. 18, when the newly mounted pipette 624′ is advancedfrom the collection tube loading station 1100, the cutter base 1158A isin the retracted position and the tip 672′ is positioned within thecutting recess 1166. Subsequently, the system controller commands thelinear actuator 1154 to extend the cutter base 1158A. The cutter 1158includes a cutting instrument 1170, e.g., a knife blade, fixedly coupledwith, or held to, the cutter base 1158A by a cutting instrument bracket1174. The cutting instrument is fixedly positioned such that when thelinear actuator 1154 extends the cutter base 1158A, the cuttinginstrument will sever the pipette tip 672′ thereby removing a portion ofthe tip 672′.

Referring now to FIG. 19, in various embodiments, after the sampled seedhas been deposited in a selected well 26 of a selected seed tray 18, thesystem controller advances the turntable 308 and positions the now emptyseed holder 304 at a cleaning station 1200. The cleaning station 1120 isoperable to clean and remove any residual seed sample and/or seedtreatment, e.g., sealant, from the respective seed holder 304 after thesampled seed has been conveyed to a seed tray 18 and before a new seedis oriented and placed in the seed holder 304. The cleaning stationcomprises a roller brush 1204 and a vacuum 1208. The vacuum 1208 isconnected to a vacuum source (not shown) to provide a vacuum at vacuumnozzle 1212 positioned in close proximity to the seed holder seedchannel 318 when the respective seed holder 304 is advanced to thecleaning station 1200. The provided vacuum will remove any residualsample material and/or seed treatment that may have collected on theseed holder 304. Additionally, the roller brush 1204 is driven, e.g.,electrically or pneumatically, to rotate on or with a roller shaft 1216.Simultaneous with providing the vacuum at the vacuum nozzle 1212, thesystem controller rotates the roller brush 1204 to remove any residualsample material and/or seed treatment that may have collected on theseed holder 304.

Applications

The present disclosure provides methods for analyzing seeds having adesired trait, marker or genotype. In one aspect of the disclosure, theanalytical methods allow individual seeds to be analyzed that arepresent in a batch or a bulk population of seeds such that the chemicaland/or genetic characteristics of the individual seeds can bedetermined.

Samples prepared by the present disclosure can be used for determining awide variety of physical, morphological, chemical and/or genetic traits.Generally, such traits are determined by screening the samples for oneor more chemical or genetic characteristics indicative of the traits.Non-limiting examples of chemical characteristics include proteins,oils, starches, fatty acids, and metabolites. Accordingly, non-limitingexamples of chemical traits include protein content, starch content, oilcontent, determination of fatty acid profiles, determination ofmetabolite profiles, etc. Genetic characteristics may include, forexample, genetic markers, alleles of genetic markers, genes, DNA-derivedsequences, RNA-derived sequences, promoters, quantative trait loci(QTL), 5′UTR, 3′UTR, satellite markers, transgenes, mRNA, ds mRNA,transcriptional profiles and methylation patterns.

In some embodiments, the methods and devices of the present disclosurecan be used in a breeding program to select plants or seeds having adesired trait or marker genotype. The methods of the present disclosurecan be used in combination with any breeding methodology and can be usedto select a single generation or to select multiple generations. Thechoice of breeding method depends on the mode of plant reproduction, theheritability of the trait(s) being improved, and the type of cultivarused commercially (e.g., F₁ hybrid cultivar, pureline cultivar, etc.).Selected, non-limiting approaches for breeding the plants of the presentdisclosure are set forth below. It is further understood that anycommercial and non-commercial cultivars can be utilized in a breedingprogram. Factors such as, for example, emergence vigor, vegetativevigor, stress tolerance, disease resistance, branching, flowering, seedset, seed size, seed density, standability, and threshability etc., willgenerally dictate the choice.

In various embodiments, the methods of the present disclosure are usedto determine the genetic characteristics of seeds in a marker-assistedbreeding program. Such methods allow for improved marker-assistedbreeding programs wherein nondestructive direct seed sampling can beconducted while maintaining the identity of individuals from the seedsampler to the field. As a result, the marker-assisted breeding programresults in a “high-throughput” platform wherein a population of seedshaving a desired trait, marker or genotype can be more effectivelybulked in a shorter period of time, with less field and labor resourcesrequired. Such advantages will be more fully described below.

In other embodiments, the present disclosure provides a method foranalyzing individual seeds within a population of seeds having geneticdifferences. The method comprises removing a sample comprising cellswith DNA from seeds in the population without affecting the germinationviability of the seeds; screening the DNA extracted from the sample forthe presence or absence of at least one genetic marker; selecting seedsfrom the population based upon the results of the DNA screening; andcultivating plants from the selected seed.

As described above, the sampling systems and methods of this disclosureprotect germination viability of the seeds so as to be non-destructive.Germination viability means that a predominant number of sampled seeds(i.e., greater than 50% of all sampled seeds) remain viable aftersampling. In some particular embodiments, at least about 75% of sampledseeds, and in some embodiments at least about 85% of sampled seedsremain viable. It should be noted that lower rates of germinationviability may be tolerable under certain circumstances or for certainapplications, for example, as genotyping costs decrease with timebecause a greater number of seeds could be sampled for the same genotypecost.

In yet other embodiments, germination viability is maintained for atleast about six months after sampling to ensure that the sampled seedwill be viable until it reaches the field for planting. In someparticular embodiments, the methods of the present disclosure furthercomprise treating the sampled seeds to maintain germination viability.Such treatment may generally include any means known in the art forprotecting a seed from environmental conditions while in storage ortransport. For example, in some embodiments, the sampled seeds may betreated with a polymer and/or a fungicide to protect the sampled seedwhile in storage or in transport to the field before planting.

In various embodiments, the samples of the present disclosure are usedin a high-throughput, non-destructive method for analyzing individualseeds in a population of seeds. The method comprises removing a samplefrom the seed while preserving the germination viability of the seed;and screening the sample for the presence or absence of one or morecharacteristics indicative of a genetic or chemical trait. The methodmay further comprise selecting seeds from the population based on theresults of the screening; and cultivating plants from the selected seed.

DNA may be extracted from the sample using any DNA extraction methodsknown to those of skill in the art which will provide sufficient DNAyield, DNA quality, and PCR response. A non-limiting example of suitableDNA-extraction methods is SDS-based extraction with centrifugation. Inaddition, the extracted DNA may be amplified after extraction using anyamplification method known to those skilled in the art. For example, onesuitable amplification method is the GenomiPhi® DNA amplification prepfrom Amersham Biosciences.

The extracted DNA is screened for the presence or absence of a suitablegenetic marker. A wide variety of genetic markers are available andknown to those of skill in the art. The DNA screening for the presenceor absence of the genetic marker can be used for the selection of seedsin a breeding population. The screening may be used to select for QTL,alleles, or genomic regions (haplotypes). The alleles, QTL, orhaplotypes to be selected for can be identified using newer techniquesof molecular biology with modifications of classical breedingstrategies.

In other various embodiments, the seed is selected based on the presenceor absence of a genetic marker that is genetically linked with a QTL.Examples of QTLs which are often of interest include but are not limitedto yield, lodging resistance, height, maturity, disease resistance, pestresistance, resistance to nutrient deficiency, grain composition,herbicide tolerance, fatty acid content, protein or carbohydratemetabolism, increased oil content, increased nutritional content, stresstolerance, organoleptic properties, morphological characteristics, otheragronomic traits, traits for industrial uses, traits for improvedconsumer appeal, and a combination of traits as a multiple trait index.Alternatively, the seed can be selected based on the presence or absenceof a marker that is genetically linked with a haplotype associated witha QTL. Examples of such QTL may again include, without limitation,yield, lodging resistance, height, maturity, disease resistance, pestresistance, resistance to nutrient deficiency, grain composition,herbicide tolerance, fatty acid content, protein or carbohydratemetabolism, increased oil content, increased nutritional content, stresstolerance, organoleptic properties, morphological characteristics, otheragronomic traits, traits for industrial uses, traits for improvedconsumer appeal, and a combination of traits as a multiple trait index.

Selection of a breeding population could be initiated as early as the F₂breeding level, if homozygous inbred parents are used in the initialbreeding cross. An F₁ generation could also be sampled and advanced ifone or more of the parents of the cross are heterozygous for the allelesor markers of interest. The breeder may screen an F₂ population toretrieve the marker genotype of every individual in the population.Initial population sizes, limited only by the number of available seedsfor screening, can be adjusted to meet the desired probability ofsuccessfully identifying the desired number of individuals. See Sedcole,J. R. “Number of plants necessary to recover a trait.” Crop Sci.17:667-68 (1977). Accordingly, the probability of finding the desiredgenotype, the initial population size, and the targeted resultingpopulation size can be modified for various breeding methodologies andinbreeding level of the sampled population.

The selected seeds may be bulked or kept separate depending on thebreeding methodology and target. For example, when a breeder isscreening an F₂ population for disease resistance, all individuals withthe desired genotype may be bulked and planted in the breeding nursery.Conversely, if multiple QTL with varying effects for a trait such asgrain yield are being selected from a given population, the breeder maykeep individual identity preserved, going to the field to differentiateindividuals with various combinations of the target QTL.

Several methods of preserving single seed identity can be used whiletransferring seed from the chipping lab to the field. Methods include,but are not limited to, transferring selected individuals to seed tape,a cassette tray, or indexing tray, transplanting with peat pots, andhand-planting from individual seed packets. Multiple cycles of selectioncan be utilized depending on breeding targets and genetic complexity.

The screening methods of the disclosure may further be used in abreeding program for introgressing a trait into a plant. Such methodscomprise removing a sample comprising cells with DNA from seeds in apopulation, screening the DNA extracted from each seed for the presenceor absence of at least one genetic marker, selecting seeds from thepopulation based upon the results of the DNA screening; cultivating afertile plant from the seed; and utilizing the fertile plant as either afemale parent or male parent in a cross with another plant.

Examples of genetic screening to select seeds for trait integrationinclude, without limitation, identification of high recurrent parentallele frequencies, tracking of transgenes of interest or screening forthe absence of unwanted transgenes, selection of hybrid testing seed,and zygosity testing.

The identification of high recurrent pair allele frequencies via thescreening methods of the present disclosure again allows for a reducednumber of rows per population and an increased number of populations, orinbred lines, to be planted in a given field unit. Thus, the screeningmethods of the present disclosure may also effectively reduce theresources required to complete the conversion of inbred lines.

The methods of the present disclosure further provide quality assurance(QA) and quality control by assuring that regulated or unwantedtransgenes are identified and discarded prior to planting.

The methods of the present disclosure may be further applied to identifyhybrid seed for transgene testing. For example, in a conversion of aninbred line at the BCnF₁ stage, a breeder could effectively create ahybrid seed lot (barring gamete selection) that was 50% hemizygous forthe trait of interest and 50% homozygous for the lack of the trait inorder to generate hybrid seed for testing. The breeder could then screenall F₁ seeds produced in the test cross and identify and select thoseseeds that were hemizygous. Such method is advantageous in thatinferences from the hybrid trials would represent commercial hybridgenetics with regard to trait zygosity.

Other applications of the screening methods of this disclosure foridentifying and tracking traits of interest carry the same advantagesidentified above with respect to required field and labor resources.Generally, transgenic conversion programs are executed in multi-seasonlocations which carry a much higher land and management cost structure.As such, the impact of either reducing the row needs per population orincreasing the number of populations within a given field unit aresignificantly more dramatic on a cost basis versus temperateapplications.

Still further, the screening methods of this disclosure may be used toimprove the efficiency of the doubled haploid program through selectionof desired genotypes at the haploid stage and identification of ploidylevel to eliminate non-haploid seeds from being processed and advancingto the field. Both applications again result in the reduction of fieldresources per population and the capability to evaluate a larger numberof populations within a given field unit.

In various embodiments, the disclosure further provides an assay forpredicting embryo zygosity for a particular gene of interest (GOI). Theassay predicts embryo zygosity based on the ratio of the relative copynumbers of a GOI and of an internal control (IC) gene per cell or pergenome. Generally, this assay uses an IC gene that is of known zygosity,e.g., homozygous at the locus (two IC copies per diploid cell), fornormalizing measurement of the GOI. The ratio of the relative copynumbers of the IC to the GOI predicts the GOI copy number in the cell.In a homozygous cell, for any given gene (or unique genetic sequence),the gene copy number is equal to the cell's ploidy level since thesequence is present at the same locus in all homologous chromosomes.When a cell is heterozygous for a particular gene, the gene copy numberwill be lower than the cell's ploidy level. The zygosity of a cell atany locus can thus be determined by the gene copy number in the cell.

In some particular embodiments, the disclosure provides an assay forpredicting corn embryo zygosity. In corn seed, the endosperm tissue istriploid, whereas the embryo tissue is diploid. Endosperm that ishomozygous for the IC will contain three IC copies. Endosperm GOI copynumber can range from 0 (homozygous negative) to 3 (homozygouspositive); and endosperm GOI copy number of 1 or 2 is found in seedheterozygous for the GOI (or hemizygous for the GOI if the GOI is atransgene). Endosperm copy number is reflective of the zygosity of theembryo: a homozygous (positive or negative) endosperm accompanies ahomozygous embryo, heterozygous endosperm (whether a GOI copy number of1 or 2) reflects a heterozygous (GOI copy number of 1) embryo. Theendosperm GOI copy number (which can range from 0 to 3 copies) can bedetermined from the ratio of endosperm IC copy number to endosperm GOIcopy number (which can range from 0/3 to 3/3, that is, from 0 to 1),which can then be used to predict zygosity of the embryo.

Copy numbers of the GOI or of the IC can be determined by any convenientassay technique for quantification of copy numbers, as is known in theart. Examples of suitable assays include, but are not limited to, RealTime (TaqMan®) PCR (Applied Biosystems, Foster City, Calif.) andInvader® (Third Wave Technologies, Madison, Wis.) assays. Preferably,such assays are developed in such a way that the amplificationefficiency of both the IC and GOI sequences are equal or very similar.For example, in a Real Time TaqMan® PCR assay, the signal from asingle-copy GOI (the source cell is determined to be heterozygous forthe GOI) will be detected one amplification cycle later than the signalfrom a two-copy IC, because the amount of the GOI is half that of theIC. For the same heterozygous sample, an Invader® assay would measure aGOI/IC ratio of about 1:2 or 0.5. For a sample that is homozygous forboth the GOI and the IC, the GOI signal would be detected at the sametime as the IC signal (TaqMan®), and the Invader assay would measure aGOI/IC ratio of about 2:2 or 1.

These guidelines apply to any polyploid cell, or to haploid cells (suchas pollen cells), since the copy number of the GOI or of the IC remainproportional to the genome copy number (or ploidy level) of the cell.Thus, these zygosity assays can be performed on triploid tissues such ascorn endosperm.

The description herein is merely exemplary in nature and, thus,variations that do not depart from the gist of that which is describedare intended to be within the scope of the teachings. Such variationsare not to be regarded as a departure from the spirit and scope of theteachings.

What is claimed is:
 1. An automated seed sampler system, comprising: animaging device configured to obtain at least one image of a seed; anorienting device configured to orient the seed based on the at least oneimage of the seed; and a sampling station configured to remove tissuefrom the oriented seed.
 2. The system of claim 1, wherein the imagingdevice includes at least one camera.
 3. The system of claim 1, furthercomprising an imaging fixture configured to support the seed adjacentthe imaging device so that the imaging device can obtain the at leastone image of the seed; and wherein the orienting device is configured toorient the seed while the seed is supported within the imaging fixture.4. The system of claim 1, wherein the orienting device includes anactuator configured to orient the seed in a desired orientation.
 5. Thesystem of claim 4, wherein the actuator is selected from the groupconsisting of an air-operated actuator and a mechanical actuator.
 6. Thesystem of claim 1, further comprising a seed transport subsystemconfigured to support the oriented seed and convey the oriented seed tothe sampling station.
 7. The system of claim 6, wherein the seedtransport subsystem includes a seed holder for holding the oriented seedin the seed transport subsystem, and wherein the sampling station isconfigured to remove tissue from the oriented seed in the seed holder.8. The system of claim 1, wherein the sampling station is configured toremove tissue from the oriented seed so as to protect germinationviability of the seed.
 9. The system of claim 1, further comprising asample tray for holding the tissue removed from the oriented seed, and aseed tray for holding the seed from which the tissue is removed suchthat a one-to-one correspondence exists between the tissue and the seedfrom which the tissue is removed.
 10. A method for removing tissue froma seed, the method comprising: imaging a seed; orienting the seed basedon information obtained from imaging the seed; and removing tissue fromthe oriented seed.
 11. The method of claim 10, wherein imaging a seedincludes suspending the seed in an imaging fixture using air, andimaging the suspended seed.
 12. The method of claim 11, whereinorienting the seed includes rotating the seed in the imaging fixture toa desired orientation.
 13. The method of claim 10, wherein imaging aseed includes locating a tip portion of the seed; and wherein orientingthe seed includes orienting the seed based on the location of the tipportion of the seed.
 14. The method of claim 10, wherein orienting theseed includes orienting the seed in a desired orientation using anactuator.
 15. The method of claim 10, further comprising transportingthe oriented seed in a seed transport subsystem to a sampling stationfor removing the tissue from the oriented seed.
 16. The method of claim10, further comprising collecting the tissue removed from the orientedseed so that a one-to-one correspondence exists between the tissue andthe seed from which the tissue is removed.
 17. The method of claim 10,further comprising receiving the tissue in a receptacle and/orcontacting the tissue with extraction fluid.
 18. The method of claim 10,wherein removing tissue from the oriented seed includes removing tissuefrom the oriented seed while protecting germination viability of theseed.
 19. The method of claim 10, further comprising analyzing thetissue for one or more characteristics indicative of at least onegenetic and/or chemical trait.
 20. The method of claim 10, furthercomprising isolating the seed from a plurality of seeds prior to imagingthe seed.